This invention relates to a mammalian cDNA which encodes an RVP-1 variant and to the use of the cDNA and the encoded protein in the diagnosis and treatment of cell proliferative disorders, particularly cancers, and autoimmune disorders, particularly Crohn""s disease.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Apoptosis is the genetically controlled process by which unneeded or defective cells undergo programmed cell death. Apoptotic events are part of the normal developmental programs of many multicellular organisms. Selective elimination of cells is as important for morphogenesis and tissue remodeling as is cell proliferation and differentiation. Apoptosis is a critical component of the immune response. Cytotoxic T-cells and natural killer cells are active in systemic defense through induction of apoptosis in tumor cells and virus-infected cells. In addition, immune cells that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response.
Apoptotic cells undergo distinct morphological changes including shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology. Biochemically, apoptotic cells are characterized by increased intracellular calcium concentration, fragmentation of chromosomal DNA into nucleosomal-length units, and expression of novel cell surface components. The molecular mechanisms of apoptosis are highly conserved, and many of the key protein regulators and effectors are known. Apoptosis generally proceeds in response to a signal which is transduced intracellularly and results in altered patterns of gene expression and protein activity. Signaling molecules such as hormones and cytokines are known to regulate apoptosis both positively and negatively through their interactions with cell surface receptors. Transcription factors also play an important role in the onset of apoptosis. A number of downstream effector molecules, especially proteases, have been implicated in the process.
The rat ventral prostate is a model system for the study of hormone-regulated apoptosis. Messenger RNA transcripts including those encoding the rat protein, RVP-1, are upregulated when epithelial cells undergo apoptosis in response to androgen deprivation (Briehl and Miesfeld (1991) Mol Endocrinol 5:1381-1388). The human homolog, hRVP1, is 89% identical to the rat protein, 220 amino acids in length, and contains four transmembrane domains (Katahira et al (1997) J Biol Chem 272:26652-26658). hRVP1, also known as claudin 3 (g2459928), is highly expressed in the lung, intestine, and liver and functions as a low affinity receptor for the Clostridium perfringens enterotoxin, a causative agent of diarrhea in humans and other animals.
Claudins are a multi-gene family of four transmembrane proteins (Morita et al (1999) Proc Natl Acad Sci 96:511-516) which have been found to form tight junctions (TJs). For example, claudin 1 is a protein of 220 residues that incorporates into TJ strands. TJs form a belt-like network of strands within plasma membranes at the most apical region of polarized epithelial and endothelial cells. This network creates a permeability barrier to the lateral diffusion of lipids and proteins between apical and basolateral membrane domains and maintains cellular polarity. In the region between adjacent cells where two opposing membranes come together, tight junction strands from each cell associate to form a paired strand. These paired strands create permeability barriers for the diffusion of solutes through the paracellular pathway. Claudins belong to a superfamily of epithelial membrane proteins (EMPs) known to carry out functions in cell growth, differentiation, and apoptosis (Lobsiger et al (1996) Genomics 36:379-387). Aberrant expression of EMPs has been associated with tumorigenesis (Ben-Porath and Benvenisty (1996) Gene 183:69-75).
Many gastrointestinal disorders, including peptic ulcers, gastritis, ulcerative colitis and Crohn""s disease, are characterized by the same symptoms. These symptoms include severe abdominal pain, cramps, fever, diarrhea, anemia, and weight loss. After establishing that the symptoms are not due to a infectious agent, it becomes necessary to diagnose them in order to provide appropriate treatment. Crohn""s disease which is an autoimmune disorder appears to enter an acute phase in response to stress and the ulcerous lesions are particularly active in forming adhesions that must be removed surgically. At the present time, there are no specific therapies for this disorder.
The discovery of a mammalian cDNA encoding an RVP-1 variant and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis and treatment of cell proliferative disorders, particularly cancers, and autoimmune disorders, particularly Crohn""s disease.
The invention is based on the discovery of a mammalian cDNA which encodes a mammalian RVP-1 variant (MAPOP-1) which is useful in the diagnosis and treatment of cell proliferative disorders, particularly cancers, and autoimmune disorders, particularly Crohn""s disease.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding a mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 85% identity to the amino acid sequence of SEQ ID NO: 1, an antigenic epitope of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides an isolated mammalian cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:2, a fragment selected from SEQ ID NOs:3-7, a variant selected from SEQ ID NOs:8-11 and having at least 80% identity to the nucleic acid sequence of SEQ ID NO:2, or an oligonucleotide of SEQ ID NO:2. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding the protein having the amino acid sequence of SEQ ID NO:1. The invention further provides a vector containing the cDNA, a host cell containing the vector, and a method for using the cDNA to produce MAPOP-1 comprising culturing the host cell under conditions for protein expression and recovering the protein from cell culture. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding an MAPOP-1. The invention additionally provides a mammalian fragment, or the complement thereof, selected from the group consisting of SEQ ID NOs:8-11. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose a cell proliferative disorder, particularly cancer, or an autoimmune disorder, particularly Crohn""s disease. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 85% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the MAPOP-1 to treat a subject with Crohn""s disease comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with Crohn""s disease.
The invention provides a method of using a mammalian protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of Crohn""s disease.
The invention also provides a method of using a mammalian protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which binds specifically to MAPOP-1. The invention also provides a method of using an antibody to diagnose Crohn""s disease comprising combining the antibody with a sample under conditions to allow specific binding, detecting the antibody complex, and comparing the quantity of antibody complex to known standards, thereby establishing the presence of Crohn""s disease. The invention further provides a method of using an antibody to treat Crohn""s disease comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:3-11, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.